Aging is a complex process that influences many aspects of our lives yet little is known about the molecular pathways that regulate this process. Calorie Restriction (CR) extends life span in a wide sprectrum of organisms. Understanding the mechanism by which CR extends life span will help to elucidate the molecular pathways of longevity regulations. The main goal of this proposal is to utilize the genetically tractable yeast Saccharomyces cerevisiae as a model system to identify and unravel the longevity regulating pathways. The major hypothesis of this research is that longevity factors are subject to metabolic regulation under Calorie Restriction (CR) and the intracellular NAD/NADH homeostasis factors play an important role in mediating this regulation. Aim 1): To understand the role of metabolic enzymes in longevity regulation. Using Affinity-purification, molecular and genetic studies, we will elucidate how metabolic enzymes regulate longevity and to identify other longevity regulating metabolic factors. Aim 2): To study the role of NAD/NADH homeostasis in the metabolic regulation of longevity. In vivo levels of NAD and NADH will be determined using Nano-HPLC/AMS analysis. The mechanisms by which the NADINADH homeostasis factors regulate life span will be studied. Aim 3): To identify new components in the longevity signaling pathway. In this Specific Aim, we propose three different approaches that combine genetic, genomic and biochemical methods to detail the pathways of CR and to identify new longevity pathways. Many cellular pathways are conserved in yeast and mammals. Therefore, insight into these molecular processes in yeast will also provide clues to the molecular basis of human aging and age-associated diseases.